This is a proposal for continued support to evaluate membrane transport, metabolism, and binding of folates and antifolates in rat hepatocytes. Initial studies indicate complex interactions between the antifolate, methotrexate (MTX), and freshly isolated hepatocytes consisting of (1) MTX transport across the cell membrane, (2) MTX binding to dihydrofolate reductase, (3) MTX binding to additional high-affinity site(s), (4) the accumulation of an exchangeable intracellular MTX component that reaches steady-state at a level greater than that predicted for a passive system, (5) rapid polyglutamation of MTX. The initial uptake phase is in part saturable, and is temperature and sulfhydryl-group dependent. There is additional evidence for energy-dependent transport in that MTX uptake is reduced by metabolic inhibitors and removal of extracellular Na ion. The proposed studies will evaluate MTX and 5-CH3-H4-folate interactions with hepatocytes in freshly isolated cells as well as hepatocytes in monolayer culture. (1) The initial uptake process will be characterized using rapid sampling techniques to clearly identify the influx phase. (2) Energetics of transport will be assessed including a possible role of cyclic nucleotides in the transport process. (3) Compartmental analysis of exchangeable intracellular folates will be evaluated in an attempt to distinguish between free and loosely bound components within the cell. (4) Studies will attempt to isolate macromolecules which bind MTX and/or 5-CH3-H4-folate. (5) The capacity of hepatocytes to rapidly polyglutamate MTX will be utilized as a model to characterize the properties of polyglutamate synthesis and the physical state of these derivatives within the cell. (6) Studies will characterize the pharmacologic effects of cytotoxicity of MTX in hepatocytes. (7) Finally, studies will assess the characteristics of liposomal interactions with hepatocytes, the effects of this interaction on transport of folate compounds across the cell membrane, the effects of liposomal encapsulation of folates on their delivery into the cell.